Abstract:

Campesterol, as an important synthetic precursor of steroid drugs (progesterone, androstenedione, hydrocortisone, etc.), has received extensive attention from domestic and foreign researchers. First, through bioinformatics analysis, 10 different sources of 7-dehydrocholesterol reductase DHCR7 encoding gene DHCR7 were isolated, and the endogenous ERG5 gene of Saccharomyces cerevisiae was inactivated by using the CRISPR/Cas9 gene editing technology to construct the recombinant strain for producing campesterol. The results showed that the Zw507, integrated Pangasianodon hypophthalmus DHCR7, obtains the yield of campesterol 216.93 mg/L. Then, 10 yeast endogenous strong promoters were selected to combine with PhDHCR7. The results showed that when TEF1p was used as the promoter of PhDHCR7, the production of campesterol was up to 253.35 mg/L. In order to further increase the output of campesterol, the copy number of the PhDHCR7 expression cassette in the yeast genome was increased. When the copy number is 3, the output of campesterol reaches the highest 302.27 mg/L. Finally, fed-batch fermentation was carried out in a 5 L fermentor, and the yield of campesterol was 916.88 mg/L. Thus this strain can be used as an excellent chassis cell for subsequent biosynthesis of steroidal drugs.

Key words: 7-dehydrocholesterol reductase, Saccharomyces cerevisiae, campesterol, synthetic biology, fed-batch fermentation

摘要：

菜油甾醇作为甾体药物（孕酮、雄烯二酮、氢化可的松等）的重要合成前体已受到国内外研究学者的广泛关注。首先通过生物信息学分析，筛选了10种不同来源的7-脱氢胆固醇还原酶DHCR7，并采用CRISPR/Cas9基因编辑技术将酿酒酵母（Saccharomyces cerevisiae）内源的ERG5基因替换成不同来源的DHCR7基因，构建了菜油甾醇合成菌株。结果发现整合来源于Pangasianodon hypophthalmus DHCR7的菌株Zw507表现出最高的菜油甾醇的产量216.93 mg/L。进一步筛选了10种酵母内源启动强度较强的启动子来与PhDHCR7基因进行组合，结果显示以TEF1p为启动子时菜油甾醇的产量最高可达253.35 mg/L。为了进一步提高菜油甾醇产量，增加了DHCR7表达盒在酵母基因组上的拷贝数。当拷贝数为3个时，菜油甾醇的产量达到最高302.27 mg/L。最终，通过5 L发酵罐进行补料分批发酵，实现了916.88 mg/L菜油甾醇产量。该菌株可作为后续甾体药物生物合成的优良底盘细胞。

关键词: 7-脱氢胆固醇还原酶, 酿酒酵母, 菜油甾醇, 合成生物学, 补料分批发酵